1H MAS NMR of Donor Pancreatic Tissue is a Useful Predictor of Islet Viability Prior to Islet Isolation

AbstractA significant limitation and cost to any clinical islet program is the related to processing human pancreas and not recovering significant numbers of viable islets for clinical transplantation. The development of an assay system that could be utilized and provide an index of cell and tissue viability before islet isolation would provide a major impact on the scientific aspects of organ preservation and a huge cost saving to any clinical islet transplantation program.Metabolomic analysis by 1H MAS NMR was used to assess samples of donor pancreatic tissue taken prior to islet isolation. A significant correlation was observed between the ratio of the combined integrals of the sugar (3.5-4.5 ppm) and choline (3.0-3.5 ppm) regions to the integrals of the CH3 (0.9 ppm) and CH2 (1.3 ppm) peaks of the 1H MAS NMR spectra of pancreatic tissue samples taken prior to islet isolation and the glucose responsiveness, a measure of islet viability, of the isolated islets (P<0.05). The effect of the two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method, previously shown to restore ischemically damaged pancreases by increasing oxygenation, was also studied using 1H MAS NMR spectroscopy. PFC recovery of the donor pancreas also correlated with an increase in the combined integrals of the sugar and choline regions to the CH3 and CH2 peaks of the 1H MAS NMR spectra (P<0.05). In addition, significant differences in the integrals of the sugar region and CH2 peaks were observed between the pre- and post-PFC samples (P<0.05). These results support the notion that specific metabolites observed in 1H MAS NMR can be used as a means to assess reversible/irreversible tissue damage and offers a means to assess donor pancreatic tissue prior to islet isolation for transplantation.

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Amount and Distribution of Collagen in Pancreatic Tissue of Different Species in the Perspective of Islet Isolation Procedures

Cell Transplantation ◽

10.1177/096368979500400610 ◽

1995 ◽

Vol 4 (6) ◽

pp. 609-614 ◽

Cited By ~ 17

Author(s):

Paul T.R. van Suylichem ◽

Jan-Erik H.M. van Deijnen ◽

Gerrit H.J. Wolters ◽

Reinout van Schilfgaarde

Keyword(s):

Colorimetric Method ◽

Pancreatic Tissue ◽

Collagen Content ◽

Human Pancreas ◽

Islet Isolation ◽

Secondary Importance ◽

Enzymatic Dissociation ◽

Tissue Sections ◽

Dog Pancreas ◽

General Experience

Because collagen is the major target in the enzymatic dissociation of the pancreas for islet isolation, we determined the amount of collagen and its distribution in a comparative study comprising normal pancreata of rat, dog, man, young pig, and adult pig. Collagen content was determined using a colorimetric method and its distribution was assessed in tissue sections stained with Sirius red. The collagen content is relatively low in the rat and adult pig pancreas, and the amount of collagen is relatively low in the septa of the rat and dog pancreas. Not the amount of collagen in the septa but collagen in the rest of the pancreas, mainly located between the acini, seems to determine the dissociation of the pancreatic tissue. This can be exemplified by the higher islet yields obtained from the adult vs. the young pig pancreas; the latter contains a higher total amount of collagen but a similar, relatively high, amount of collagen in the septa. A high amount of collagen surrounding the islets seems to be of secondary importance in islet isolations, because yields of the same magnitude are obtained from the canine and human pancreas containing a relatively low vs. high amount of collagen around the islets but a similar total collagen content. The rat pancreas contains both a low total amount of collagen and a high amount of collagen around the islets; therefore, the general experience that islet isolation procedures are effective in rats can be readily understood.

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The Effect of UW Solution and Its Components on the Collagenase Digestion of Human and Porcine Pancreas

Cell Transplantation ◽

10.1177/096368979500400611 ◽

1995 ◽

Vol 4 (6) ◽

pp. 615-619 ◽

Cited By ~ 27

Author(s):

Harold H. Contractor ◽

Paul R. V. Johnson ◽

David R. Chadwick ◽

Gavin S. M. Robertson ◽

Nicholas J. M. London

Keyword(s):

Cold Storage ◽

Species Difference ◽

Human Pancreas ◽

Islet Isolation ◽

Porcine Pancreas ◽

Uw Solution ◽

Porcine Islet ◽

University Of Wisconsin ◽

Collagenase Digestion ◽

Tissue Dissociation

University of Wisconsin (UW) solution is used extensively as a cold storage solution during the procurement and transport of the pancreas prior to islet isolation. However, it has been observed that UW inhibits the collagenase digestion phase of human but not porcine islet isolation, resulting in poor islet yields and islets of poor viability. The aim of this study was, therefore, to confirm this species difference and to determine which components of UW are responsible for the inhibition in the human. In the initial experiment, blocks of human and porcine pancreas (n = 7) were incubated in test tubes containing collagenase at a concentration of 4 mg/mL at 37°C dissolved in 4 mL of either Hanks' solution or UW. Every 5 min the tubes were manually shaken and the degree of tissue dissociation scored on a scale of + and +++. Our results confirm the inhibition of collagenase digestion in the human but not the pig. Using the same methodology, we then investigated the components of UW that were causing the observed inhibition in the human pancreas (n = 7). This time the collagenase was dissolved in individual or combinations of UW components. Using Hank's as a control, the results were then expressed as a median ratio. The components found to be most inhibitory were magnesium, the Na+/K+ ratio, hydroxyethyl starch (HES), and adenosine. Allopurinol in combination with either lactobionate or glutathione was markedly inhibitory (i.e., median ratio 1.8 and 1.9, respectively). The most inhibitory solution tested was a combination of the three components raffinose, glutathione, and lactobionate (median ratio 2.1). This combination was almost as inhibitory as UW itself (median ratio 2.7). These findings are essential for the development of effective cold-storage solutions for the human pancreas that do not inhibit the subsequent collagenase digestion phase of islet isolation.

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1H-MAS-NMR Chemical Shifts in Hydrogen-Bonded Complexes of Chlorophenols (Pentachlorophenol, 2,4,6-Trichlorophenol, 2,6-Dichlorophenol, 3,5-Dichlorophenol, and p-Chlorophenol) and Amine, and H/D Isotope Effects on 1H-MAS-NMR Spectra

Molecules ◽

10.3390/molecules18044786 ◽

2013 ◽

Vol 18 (4) ◽

pp. 4786-4802 ◽

Cited By ~ 4

Author(s):

Hisashi Honda

Keyword(s):

Nmr Spectra ◽

Chemical Shifts ◽

Isotope Effects ◽

Mas Nmr ◽

1H Mas Nmr ◽

Nmr Chemical Shifts ◽

Bonded Complexes ◽

Hydrogen Bonded

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Calcium

Cell Transplantation ◽

10.1177/0963689718779350 ◽

2018 ◽

Vol 27 (7) ◽

pp. 1031-1038 ◽

Cited By ~ 2

Author(s):

Torsten Eich ◽

Magnus Ståhle ◽

Bengt Gustafsson ◽

Rune Horneland ◽

Marko Lempinen ◽

...

Keyword(s):

Enzyme Activity ◽

Islet Transplantation ◽

Clinical Chemistry ◽

Pancreatic Enzyme ◽

Human Islets ◽

Human Pancreas ◽

Islet Isolation ◽

Clinical Islet Transplantation ◽

The Media ◽

Major Reduction

Background: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5–10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. Methods: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. Results: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. Conclusions: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

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Comparison of Trypsin Inhibitors in Preservation Solution for Islet Isolation

Cell Transplantation ◽

10.1177/096368970901805-609 ◽

2009 ◽

Vol 18 (5-6) ◽

pp. 541-548 ◽

Cited By ~ 7

Author(s):

Hirofumi Noguchi ◽

Michiko Ueda ◽

Shuji Hayashi ◽

Naoya Kobayashi ◽

Teru Okitsu ◽

...

Keyword(s):

Islet Transplantation ◽

Stimulation Index ◽

Islet Isolation ◽

Preservation Solution ◽

University Of Wisconsin ◽

Trypsin Inhibition ◽

Clinical Islet Transplantation ◽

Pancreas Preservation ◽

Collagenase Inhibition

Islet transplantation has recently emerged as an effective therapy and potential cure for type 1 diabetes mellitus. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. Moreover, we recently reported that islet yield was significantly higher in the ET-Kyoto solution with ulinastatin (MK)/PFC preservation solution compared with the UW/PFC preservation solution in the porcine model and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with another trypsin inhibitor, Pefabloc, in preservation solution for islet isolation. Islet yield before purification was higher in the MK/PFC group compared with the ET-Kyoto with Pefabloc (PK)/PFC group. The stimulation index was higher for the MK/PFC group than for the PK/PFC group. These data suggest that ET-Kyoto with ulinastatin was the better combination for pancreas preservation than ET-Kyoto with Pefabloc. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

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PROLONGED PRESERVATION OF THE HUMAN PANCREAS PRIOR TO ISLET ISOLATION USING THE TWO-LAYER (UNIVERSITY OF WISCONSIN SOLUTION [UW]/ PERFLUOROCARBON) METHOD.

Transplantation Journal ◽

10.1097/00007890-200004271-00384 ◽

2000 ◽

Vol 69 (Supplement) ◽

pp. S213 ◽

Cited By ~ 5

Author(s):

Shinichi Matsumoto ◽

Sabrina A. Qualley ◽

Theodore H. Rigley ◽

Christopher L. Marsh ◽

Rick B. Stevens

Keyword(s):

Human Pancreas ◽

Islet Isolation ◽

University Of Wisconsin Solution ◽

University Of Wisconsin ◽

Wisconsin Solution

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High Aluminum Ordering in SSZ-59: Residual 1H–27Al Dipolar Coupling Effects in 1H MAS NMR Spectra of Brønsted Acid Sites in Zeolites

The Journal of Physical Chemistry C ◽

10.1021/acs.jpcc.0c11379 ◽

2021 ◽

Vol 125 (8) ◽

pp. 4869-4877

Author(s):

Christian Schroeder ◽

Stacey I. Zones ◽

Christian Mück-Lichtenfeld ◽

Michael Ryan Hansen ◽

Hubert Koller

Keyword(s):

Nmr Spectra ◽

Dipolar Coupling ◽

Acid Sites ◽

Mas Nmr ◽

Bronsted Acid ◽

Coupling Effects ◽

Brønsted Acid Sites ◽

1H Mas Nmr ◽

Brönsted Acid ◽

High Aluminum

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High-resolution 1H MAS NMR spectra of 2∶1 phyllosilicates

Chemical Communications ◽

10.1039/a906577f ◽

2000 ◽

pp. 37-38 ◽

Cited By ~ 20

Author(s):

María D. Alba ◽

Ana I. Becerro ◽

Miguel A. Castro ◽

Ana C. Perdigón

Keyword(s):

High Resolution ◽

Nmr Spectra ◽

Mas Nmr ◽

1H Mas Nmr

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Efficacy of the Oxygen-Charged Static Two-Layer Method for Short-Term Pancreas Preservation and Islet Isolation from Nonhuman Primate and Human Pancreata

Cell Transplantation ◽

10.3727/000000002783985332 ◽

2002 ◽

Vol 11 (8) ◽

pp. 769-777 ◽

Cited By ~ 74

Author(s):

Shinichi Matsumoto ◽

Theodore H. Rigley ◽

Sabrina A. Qualley ◽

Yoshikazu Kuroda ◽

Jo Anna Reems ◽

...

Keyword(s):

Nonhuman Primate ◽

Oxygen Supply ◽

Human Model ◽

Human Pancreas ◽

Islet Isolation ◽

Primate Model ◽

University Of Wisconsin ◽

Layer Method ◽

Pancreas Preservation

Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4–13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 ± 3619 IE/g in the static TLM, 21,895 ± 3742 IE/g in the original TLM, and 6773 ± 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 ± 549 IE/g in the static TLM, 2244 ± 557 IE/g in the original TLM, and 1293 ± 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.

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